Detection of chromosomal aneuploidy in ancient genomes

Ancient DNA is a valuable tool for investigating genetic and evolutionary history that can also provide detailed profiles of the lives of ancient individuals. In this study, we develop a generalised computational approach to detect aneuploidies (atypical autosomal and sex chromosome karyotypes) in the ancient genetic record and distinguish such karyotypes from contamination. We confirm that aneuploidies can be detected even in low-coverage genomes ( ~ 0.0001-fold), common in ancient DNA. We apply this method to ancient skeletal remains from Britain to document the first instance of mosaic Turner syndrome (45,X0/46,XX) in the ancient genetic record in an Iron Age individual sequenced to average 9-fold coverage, the earliest known incidence of an individual with a 47,XYY karyotype from the Early Medieval period, as well as individuals with Klinefelter (47,XXY) and Down syndrome (47,XY, + 21). Overall, our approach provides an accessible and automated framework allowing for the detection of individuals with aneuploidies, which extends previous binary approaches. This tool can facilitate the interpretation of burial context and living conditions, as well as elucidate past perceptions of biological sex and people with diverse biological traits.

CH163 is the partial skull of a young adult female.It had been reconstructed by the excavators prior to our examination.The calvarium is largely complete, with the exception of a missing section of the anterior left parietal.The maxillofacial region is less complete, missing the right nasal bones, both ethmoid and zygomatic bones, and the sphenoid.The right hemi-mandible is complete apart from a damaged coronoid process.The left hemimandible is missing part of the interior body and much of the ascending ramus.A large number of Wormian bones are present on the occipital.
There is a well-documented association with chronic intestinal haemorrhage 1 , in turn linked with anaemia 2 and the orbits of the C10090 cranium show marked pitting known as cribra orbitalia (Figure 3a), widely interpreted as a response to anaemia.This is usually attributed to insufficient dietary iron, but other causes can include thalassemia and high parasite loads 3 .A possible link between cribra orbitalia and Turner syndrome has also been noted in a suspected case from early-twentieth-century Portugal, though the diagnosis has not been confirmed genetically 4 .
The maxillary right canine and all molars are present apart from the right first molar.The three mandibular right molars and the left second molar are present.All other teeth have been lost post-mortem.First and second molars show moderate cusp wear with no dentine exposure.The third molars are minimally worn.
The eruption and state of wear of the teeth suggests that the skull is that of a young adult.The third molar roots are fully formed and the apex appears to be closed, though slight damage at the visible root tip makes this uncertain.This together with its minimal wear would suggest an age ca.18-22 years 5,6 .However, the spheno-occipital synchondrosis (SOS) is unfused, which would normally indicate an age of no more than 14-16 years in females [7][8][9][10] (though see 11 who note that some studies support later fusion).This age discrepancy may be another effect of Turner syndrome, in which skeletal growth (including fusion) may be delayed relative to dental development 12,13 .Furthermore, the relationship between SOS closure and the onset of puberty 7 implies that the Charterhouse Warren individual would not have experienced menarche, despite her age.A delay in, or absence of, menarche is a feature of Turner syndrome, caused by the absence or low levels of the growth hormone oestrogen 14,15 .
There is an irregular, narrow oblong perforation to the mid-frontal bone that is interpreted as post-rather than peri-mortem in origin.While there is some internal bevelling, the radiating fractures have transverse fracture margins that statistically are more often seen in dry bone 16,17 .
There is mild healed porotic hyperostosis on the posterior frontal and anterior parietal bones (stage 1 in 18 ).The roofs of both orbits show marked porosity typical of cribra orbitalia, extending onto the orbital notches (stage 4 in 18 ).

Radiocarbon dating
Author: Rick J. Schulting A sample (0.68g) of the mandible containing the tooth used in the genomic analysis (C10090) was taken for AMS (accelerator mass spectrometry) radiocarbon dating.Pre-treatment followed the standard protocols in place at the Oxford Radiocarbon Accelerator Unit in the School of Archaeology, University of Oxford 19 .The results meet the quality control criteria for well-preserved collagen 20,21   As the long bones were not fully fused, sex was assessed using the features of the pubic bone 31,32 , as these features and especially the ventral arc have shown to be reliable for adolescents 33 .Other features of the pelvis 29 were considered to support the assessment but not replied upon, due to the age of the individual.Likewise, features of the skull 24,34 were not considered, as these have not been established for older adolescents, and the crania of all young adults tend to be more gracile.All three Phenice (1969) 35 traits suggested this individual was male.This was supported by a narrow sub-pubic angle and moderately narrow greater sciatic notch.Thus, this individual was sexed (skeletally) as male.
Skeleton Wetwang Slack 267 -Lab Code C10462 -Karyotype (47,XY,+21) Wetwang Slack 267 comprises the incomplete and fragmentary remains of an infant, represented by a large number of cranial fragments and two long bone shaft fragments more likely to be from the femora, tibiae or humeri than the radii, ulnae or fibulae.The sizes suggest two different bones were present, possibly a femur and a humerus.Three dental crowns were present.It was not possible to ascertain if these were mandibular or maxillary teeth, due to the early stages of development.The canine and first molar were assessed as being between cusp outline complete and crown half complete; the second collar was assessed as being between coalescence of cusps and cusp outline complete.This provides and age estimate of below 0.2 years 36 and birth plus or minus 1 week 5 .As the latter method was developed using more fetal and neonatal individuals and included both maxillary and mandibular teeth, the younger age estimate is preferred.This suggests Wetwang Slack 267 was a neonate / full term infant.No attempt was made to sex this individual as methods of sex assessment for non-adults are generally regarded as less reliable than those for adults 38 .

1.3.The Medieval Cemetery under Longwall Quad, Magdalen College, Oxford
Authors: Helen Webb, Louise Loe Skeleton 4205 -Lab Code C11119 -Karyotype (47,XXY) Skeleton 4205, was one of 115 discrete, articulated skeletons, excavated from a 12 th -13 th century Christian cemetery by Oxford Archaeology during redevelopment of the library at Magdalen College, Oxford 31,39,40 .The skeletons were found under Longwall Quad and have been interpreted as coming from the cemetery attached to the Hospital of St John.
Like the other skeletons in the cemetery, and in keeping with Christian burial tradition, 4205 was recovered from a plain earth cut east-west orientated grave.The grave was located towards the southern limit of the cemetery and the skeleton was lying in a supine (on back) position with their arms and legs extended and their head positioned on its left side facing north.It is not possible to draw any conclusions regarding the significance of the position of the grave because only the south-west corner of the cemetery was revealed during the excavation.A small number of residual pot and animal bone fragments was recovered from the backfill of the grave, but there were no artefacts associated with the burial, nor was there any evidence for a coffin.Although no shroud pins or other evidence was found, the individual had probably been buried in a shroud or winding sheet, as was common for the period 29,31 .
Skeleton 4205 was approximately 90 percent complete with only bones from the feet missing.The bones were relatively intact, with surfaces which exhibited slight, patchy erosion that penetrated the bone surfaces in some areas (consistent with Grade 2 after 35 ).Overall, the skeleton was in good condition.
Based on sexually dimorphic traits of the skull 41 and pelvis 41 the skeleton was estimated to be male.Eight out of ten of the cranial traits were of definite male expression, while two (frontal slope and mandibular ramus width) were consistent with male features but were not as clearly expressed (these were recorded as 'probable male').Similarly, of the six observable pelvic traits (seven pelvic features were not recordable), five were definitely male and one, the ilium auricular surface, was probably male.Sexually dimorphic traits of mixed expression, as described here, are typically encountered in most skeletons from all time periods 42 .
The dimensions of the femoral (50.0mm) and humeral heads (47.5mm) were employed as secondary indicators of osteological sex (with reference to 43,44 ).Both measurements are within the male ranges 45,46 (humeral head diameter >47mm, femoral head diameter >48mm).Based on morphological changes on the pubic symphyses 47 and auricular surfaces 48,49 of the pelvis, as well as the level of molar attrition 50 , the skeleton was aged as a middle (36-45 years)  or mature adult (45+ years), possibly as young as 36 years but likely over 45 years at death.
The maximum length of the left femur (48.2cm) applied to Trotter's regression formula for white males 51 indicated that the stature of Skeleton 4205 was 176.13 cm (+/-3.27cm),or 5 ft 9.34 in.This is taller than the average male stature of 171.50cm (based on 16 males), calculated for the assemblage and the third tallest male stature in the assemblage.
Although not exceptionally tall (the skeleton does not classify as of clinically tall stature because it is not taller than 2SD above the mean for the male sample -this would be 182.4cm(SD is 5.45)), the taller than average stature may be significant in terms of the diagnosis of Klinefelter syndrome, considering one of the classic, physiological signs of the condition is taller than average height 52 .
Pathology included minor, well healed inflammatory lesions (periostitis) on the tibiae (shin bones), a well healed fracture of the fourth left metacarpal (hand bone) and lesions pertaining to joint disease in the spine (Schmorl's nodes, marginal osteophytes, spondylosis deformans and osteoarthritis) and hip joints (osteoarthritis), which are particularly common in mature adult skeletons, such as this.In addition, calculus (mineralised plaque) deposits, carious lesions and dental enamel hypoplasia (DEH) were present, each of these involving multiple teeth.The DEH, which appears as lines, pits or grooves in the dental enamel, represents an interruption of the enamel development during childhood 53 and results from prolonged periods of illness and/or malnutrition 54 .The teeth affected, and the positions of the DEH lesions, indicated that the individual had suffered, and recovered from, repeated episodes of physiological stress between the ages of three and 11 years 54 .

Radiocarbon dating
A sample of the right femur shaft was taken for AMS (accelerator mass spectrometry) radiocarbon dating.Pre-treatment followed the standard protocols in place at the AMS Facility Scottish Universities Environment Research Centre (SUERC) 55 .The results are quoted in line with the international standard known as the Trondheim Convention 56 .The results meet the quality control criteria for well-preserved collagen 55 (Supplementary Table 2).The radiocarbon determination of 857 ±28 BP yields a calibrated date range of cal AD 1200-1280 (68.2%) or cal AD 1050-1080 (0.7%); 1150-1290 (94.7%).The stable isotope data from the human bone collagen suggests that the individual may have had a marine component in their diet, and based on the stable carbon isotope (δ 13 C) value of -19.7‰ and the method of Arneborg et al. 56 using end-members of -21.0‰ (fully terrestrial) and -12.5‰ (fully marine), a 15% marine contribution (±10%) has been determined.This equates to a regional marine offset (ΔR) of 0 ± 50 years which has been used in the calibration, a mix of the IntCal13 and Marine13 calibration curves 57 .Supplementary Table 2 Trinity Burial Ground (1785-1861) was an overflow cemetery, established due to concerns that the original burial ground, attached to Holy Trinity Church (now Hull Minster), would reach full capacity 25 .At least 8,791 individuals and an extensive assemblage of artefacts and coffin fittings across an area of almost 3,000m 2 , representing approximately 40% of the burial ground, were excavated by Oxford Archaeology in 2020-2021 to allow for major infrastructure improvements.
Skeleton 30828 was estimated to be nearly complete with only a few epiphyses and bones from the hands and feet missing.Some areas had undergone post-mortem breakage, but this was largely limited to the ends of the diaphyseal shafts and the cranium.Most of the cortical surfaces were affected by slight erosion (scored as a grade 3, after McKinley 2004, 16 38 ).Overall, the skeleton was judged to be in a fair condition.
The age of the individual was assessed by observing epiphyseal fusion 25 and dental development and eruption 5 .The epiphyses suggested that the individual was between 11 and 20 years of age, probably the lower end of this range, considering most epiphyses were unfused.The stage of dental development and eruption suggested an age of between 16 and 19 years.Considering that dental development and eruption correlate more closely with chronological age than epiphyseal fusion does 25 , the likely age of the individual is 16-19 years.The general lack of epiphyseal fusion suggests delayed skeletal development, or a prolonged growth period which may have resulted in a taller than average stature, had the individual lived to attain skeletal maturity.
Relatively minor pathological conditions were present.These comprised two midline developmental abnormalities, including retention of the midline frontal suture (a metopic suture, often recorded as a non-metric trait 58 ) and a first cervical vertebra with a cleft neural arch 59 .Both abnormalities are relatively common in archaeological populations and would not have had any adverse effect on the individual in life (who probably wasn't aware of them).
In addition to developmental abnormalities, bilateral type 2 cribra orbitalia (scattered fine foramina 3 ) and bilateral cribra femoralis 60 , were also present.These conditions may refer to iron deficiency anaemia 61 , but this is not certain.The aetiology of cribrous lesions such as these has been the subject of considerable debate in recent years, and there are several possible explanations, vascular imprints being one of them 62 .In the present case, the fact that the lesions are present on both orbital roofs and the femora, could suggest that iron deficiency anaemia is a more likely cause.
Another pathological condition observed in the skeleton was unilateral maxillary sinusitis (bone inflammation in the sinuses).The lesions were healed and not active at the time of death.Maxillary sinusitis may be caused by dental disease (for example, a dental abscess) or by irritants (e.g.smoke/pollution) in the environment 63 , the latter being the most likely explanation for the lesions in the present skeleton.
Lastly, all of the first molars and the right mandibular second molar had medium to large carious lesions.

1.5.Lincoln Eastern Bypass, Lincoln
Authors: Katie Keefe, Paola Ponce, Jordi Ruiz Ventura and Malin Holst Skeleton 16586 (135) -Lab Code C13582 -Karyotype (47,XYY) A total of 734 skeletons were excavated by Network Archaeology from 2016 to 2019 prior to the construction of the Lincoln Eastern Bypass, likely dating from AD 680 to 890.The skeletons were buried in west-to-east orientations in supine extended positions, following Christian tradition, in earth-cut graves, with evidence of oak coffins in some graves.
Skeleton 16586 was interred at the western edge of the southwestern part of the cemetery in the standard position and alignment.
Skeleton 16586 was 90% complete (missing the left hand, both feet and most ribs), moderately fragmented, with moderate surface preservation (Grade 3, McKinley, 2004, 16 38 ).All sexing criteria of the skull, pelvis and supplementary skeletal measurements suggested the individual was male 29,31,64 .Based on the ageing criteria of the pubic symphysis and auricular surface 42,43,64 the skeleton was a mature adult, aged 46 years old or older.The individual was 184.2cm tall 47 , which was much taller than the male mean from the site at 171.1cm, and also than the male average (172cm) for the period 65 , but was within the male stature range for the cemetery.
The skeleton had a number of minor non-metric traits that were assessed following 31,58,66 .These included an ossicle in lambdoid, an accessory lesser palatine foramen, bridging of the supraorbital notch, plaque, exostosis in the trochanteric fossa and a third trochanter.
The individual had bilateral shallow, almost imperceptible fovea capitis of the femoral heads, where the ligament for the head of the femur attached, as well as an incomplete vertebral border shift at the sacro-lumbar border, indicating a developmental defect that had occurred during the first month of gestation 59 .The first sacral vertebral body was fused to the remainder of the sacrum, as were the ala, however, the vertebra retained its inferior articulating facets, which had not been integrated into the remainder of the posterior sacral surface.The second sacral vertebra likely had superior articulating facets that corresponded with the facets on the first sacral vertebra, but unfortunately, this was not recovered.
Dental enamel hypoplasia lesions were recorded in ten of the 27 teeth.The presence of lines on the crowns of teeth as seen in the dentition of this individual occurs as a result of defective formation of tooth enamel during growth 50 , following periods of physiological stress such as malnutrition or disease during the first seven years of childhood 67 .
The individual had evidence for os acromiale in the left acromion of the scapula.The lateral end of the acromion was foreshortened and flattened, with a porotic surface.The presence of os acromiale has been linked to severe stress on the rotator cuff muscles of the shoulder during growth, which in turn prevents the natural fusion of the tip of the acromion with the acromial end.The presence of os acromiale in medieval populations has been suggested to be linked to intensive archery practice from a young age 51,68,69 .However, Mann and Hunt 70 have suggested there may be a genetic component to the condition.
A possible healed crush fracture was observed in the body of the first lumbar vertebra; the anterior body height measured 23.7mm and the posterior body height measured 30.8mm superior-inferiorly.The inferior and superior surfaces of the body were both convex.These fractures are usually caused by landing on the feet after a fall from a height, although in elderly individuals a fall onto the bottom can also lead to a crush fracture 71 .
Schmorl's nodes were recorded in the thoracic and lumbar spine.Degenerative joint changes were observed in the thoracic, lumbar and sacral vertebrae, and in both manubrioclavicular joints.Both conditions affect the joints of the spine and extraspinal areas of the skeleton due to physical activity, occupation, workload and advancing age 51,64 .
The individual had widespread calculus concretions on 26 of 27 teeth and four caries lesions.One tooth that was lost ante-mortem, while the individual also had an impacted canine, two externally draining abscesses and moderate to severe periodontal disease.Dental calculus (dental plaque), dental infections (abscesses) and the exposure of roots of teeth due to receding gums are commonly observed in archaeological populations of all periods.These conditions have been linked to poor dental hygiene, diet and heavy wear of the teeth 50,51 .Impacted teeth can develop due to a genuine failure of the tooth to develop (congenital absence), or because the tooth develops but fails to erupt (impaction) 50 as occurred with this individual whose canine was visible in the crypt.
The skeleton was buried 10m to the southwest of a young middle adult of indeterminate sex, with a very tentative diagnosis of Klinefelter's Syndrome, based on a congenital radial head dislocation, though this may also be associated with other syndromes, including Apert syndrome, Ehlers-Danlos syndrome, Nail-Patella syndrome, Klippel-Feil syndrome and kyphosis.

Target enrichment (Capture) on 1240k positions
We have developed an adjusted version of our method, specifically for genomic data enriched on the "1240k" positions (--capture).The only difference is the use of different assignment borders based on the number of SNPs per chromosome present on the "1240k" capture array commonly used in ancient DNA studies.The "autosomal baseline" Na was defined as the sum of SNPs on chromosomes 1 through 22, Na = 1,058,144 (excluding 13, 18, 21 as above).The mean Rx for XX individuals using target enrichment data was calculated as the number of SNPs on chromosome X (Nx = 49,704) divided by Na (meanRx for XX = 0.047).The mean Rx for XY individuals was (meanRx for XX) / 2, i.e. meanRx for XY = 0.023.Thus, the classification thresholds were defined at the mid-point between those two estimates and the upper Rx limit for XX was the mid-point between meanRx for two X and meanRx for three X chromosomes (meanRx for XXX = 0.070).Regarding chromosome Y, the number of SNPs on the array (Ny = 32,670) divided by Na equals to meanRy for XY = 0.03.However, empirical Ry estimates from published data 83 had much smaller values than this theoretical Ry estimate, possibly due to the abundance of repeat regions on the Y chromosome that complicate sequence alignment 78 .Since the complete absence of chromosome Y results in Ry values close to zero, we decided to define the lower Ry limit for XY individuals to a value closer to those observed in real data (lowerRy for XY = 0.004).Our method identified the 2 reported aneuploidy cases from this study too (ALM062 (47,XXX) and CLL011(47,XXY)), exhibiting robustness both in "shotgun" and "capture" sequenced genomes.
Supplementary figure 3: Application of our method using the --capture option (optimized for libraries enriched for the 1240k SNP array) on all individuals from Villalba-Mouco et al. 2021 83 .Our method identified the sex of XX and XY individuals, as well as contaminated libraries and two individuals with aneuploidy on sex chromosomes that were also reported in the original study ((ALM062 (47,XXX) and CLL011(47,XXY)).

Table 1 .
3024,25adiocarbon determination of 2456 ± 26 BP yields a calibrated date range of 2703-2365 cal BP or cal BCE 754-416 (95.4% confidence), placing it in the Early Iron Age.The stable carbon isotope (d 13 C) value of -20.9‰ is typical of a terrestrial diet.Radiocarbon dating and associated quality control data for bone collagen from Charterhouse Warren mandible (CH163).Calibrated in OxCal v.4.4 22 using Intcal20 23 .Wetwang Slack 224 was almost complete, with some small bones missing.The skull and ribs were fragmentary, and several long bones were broken.As the long bones were not fully fused, age was estimated using epiphyseal and apophyseal fusion and dental development5,24,25.The greater trochanter of the femora and distal tibiae were in the process of fusing, suggesting an age between 14 and 16 years.Complete fusion of the lesser trochanter of the femur suggests an age of at least 16 years.The distal radii, distal ulnae, distal femora, distal tibiae and distal fibulae were all unfused, suggesting an age below 18-20 years.Combined, the fusion data suggests an age of 16-18 years.Dental radiographs showed the second molars were all complete, whereas the third molars had complete roots, but no evidence of closure of the apex.This provides an age estimate of either 17.5 years 26 or 18-19 years5.As dental development is less likely to be affected by external factors28and the AlQahtani et al method5has been shown to perform well in tests of different methods30, the slightly older age estimate of 18-19 years is most appropriate for this individual.